The mechanism by which cytoplasmically synthesized mitochondrial proteins are transported into yeast mitochondria is under investigation. Two models are considered in these studies: the first is a strictly co-translational transport involving cytoplasmic ribosomes bound to the outer mitochondrial membrane, and the second is a post-translational transport process. Recent data indicate that these two processes are not mutually exclusive in the transport of proteins into yeast mitochondria. Experiments will be undertaken to assess the relative importance of each to mitochondrial biogenesis. In vitro reconstruction systems will be employed to compare the rate and extent of co-translational and post-translational transport of proteins into isolated mitochondria. These studies will then be extended to in vivo estimates of the predominant mode of transport from pulse chase studies and pool size determinations. The distribution of precursor forms of cytoplasmically synthesized mitochondrial protein will be evaluated within the context of post-translational versus co-translational transport.